DENATURATION OF A RECOMBINANT CUTINASE FROM FUSARIUM-SOLANI IN AOT-ISO-OCTANE REVERSE MICELLES - A STEADY-STATE FLUORESCENCE STUDY

Near UV absorbance and fluorescence spectroscopy show conformational c hanges of a recombinant cutinase from Fusarium solani incorporated in sodium-di-2-ethylhexyl sulfosuccinate (AOT)-iso-octane reversed micell es with W-0 = [H2O]/[AOT] = 20. Excitation spectra were used to decomp ose cutinase absorbance in its Trp and Tyr components, showing that th e latter absorb red-shifted in the native cutinase in aqueous solution as compared to free Tyr, whereas in reverse micelles and denatured cu tinase no shift is detected. Emission maxima variations (lambda(max) 3 03, 311 and 335 nm, respectively in aqueous, reverse micelles and ther mally denatured cutinase) reflect progressive changes in the micropola rity of the environment and exposure of Trp residues at the protein su rface. The encapsulation of cutinase in AOT-iso-octane reversed micell es induces a time-dependent denaturation measured by fluorescence inte nsity changes at 330 nm, which match the profile of enzyme activity lo ss in this media.